Synthetic bile acid compositions and methods

ABSTRACT

Bile acids and related compositions and methods of synthesis and use. More specifically, deoxycholic acid and related compositions, said compositions being free of all moieties of animal origin and free of pyrogenic moieties.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/472,135, filed Mar. 28, 2017, which is a continuation of U.S.application Ser. No. 14/732,515, filed Jun. 5, 2015, which is acontinuation of U.S. application Ser. No. 13/914,510, filed Jun. 10,2013, now U.S. Pat. No. 9,050,349; which is a continuation of U.S.application Ser. No. 13/486,955, filed Jun. 1, 2012, now U.S. Pat. No.8,461,140; which is a continuation of U.S. application Ser. No.12/541,045, filed Aug. 13, 2009, now U.S. Pat. No. 8,242,294; which is acontinuation-in-part of U.S. application Ser. No. 12/035,339 filed Feb.21, 2008, now abandoned; which in turn claims the benefit under 35U.S.C. 119(e) to provisional applications U.S. Application No.60/945,035 filed Jun. 19, 2007 and U.S. Application No. 60/956,875 filedAug. 20, 2007. The contents of each of the prior applications areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates broadly to bile acids and relatedcompositions and methods. In one aspect, the present invention relatesto deoxycholic acid and related compositions, useful intermediates, andmethods for synthesis thereof. In another aspect, the present inventionrelates to use of the present compositions and methods as pharmaceuticalcompositions as well as methods for the manufacture thereof.Importantly, the bile acids of the present invention are not isolatedfrom mammalian and microbial organisms naturally producing these acidsand thus are free of any toxins and contaminants associated with suchorganisms.

BACKGROUND OF THE INVENTION

Cholanology, the study of bile acids, and particularly bile acidchemistry has been of interest for the better part of a century.Although much is known, bile acid chemistry involves a wide variety ofchemical entities, many with surprising properties. For a review, see,e.g., Mukhopadhyay, S. and U. Maitra., Current Science 87: 1666-1683(2004) (“Chemistry and biology of bile acids”), incorporated herein byreference.

Bile acids are characterized by two connecting units, a rigid steroidnucleus and a short aliphatic side chain (see FIG. 1 of the presentapplication). See, Hofmann, A. F., et al. For a proposed nomenclaturefor bile acids, see J. Lipid Res. 33:599-604 (1992). Both the nucleusand the side chain have a large number of possible steric arrangements.The nucleus can be altered by expansion or contraction of individualrings, and the side chain can be shortened or lengthened. In addition,both parts of the bile acid molecule have a large number of possiblepolar substituents. Ionizing groups may be present on the nucleus or theside chain. Finally, conjugating groups may be present on the nucleus(e.g., sulfate, glucuronate, phosphate) or on the side chain (glycine ortaurine or other amino acids, or even sugars). The side chain structuredetermines the class of the compound (bile acids or bile salts).

Bile acids are amphiphiles, having both an amphiphilic and amphipathic“face”.

By convention, the hydrophobic surface is called the “β-face” and thehydrophilic surface is called the “α-face”. The β-face is lipid solubleand the α-face is relatively polar, in general. There are bile acids,such as those having polar groups (hydroxyl groups, in naturallyoccurring bile acids) on the hydrophobic face as well as on thehydrophilic face, e.g., ursodeoxycholic acid. The amphipathic nature ofthe molecule is responsible for its forming mixed micelles withamphipathic but water-insoluble lipids, such as phosphatidylcholine.Bile acids will not solubilize dietary lipids in the form of mixedmicelles unless bile acids are above a critical concentration, termedthe critical micellization concentration.

The bile acids found in greatest proportion in humans arechenodeoxycholic acid and deoxycholic acid. Deoxycholic acid is alsoknown as deoxycholate, cholanoic acid, and3α,12α-dihydroxy-5β-cholanate. In the human body deoxycholic acid isused in the emulsification of fats for the absorption in the intestine.In research, deoxycholic acid is used as a mild detergent for theisolation of membrane associated proteins. When substantially pure,deoxycholic acid is a white to off-white crystalline powder form.Deoxycholic acid is one of the four main acids produced by the liver. Itis soluble in alcohol and acetic acid. The CAS number for deoxycholicacid is [83-44-3].

Rapid removal of body fat is an age-old ideal, and many substances havebeen claimed to accomplish such results, although few have shownresults. “Mesotherapy”, or the use of injectables for the removal offat, is not widely accepted among medical practitioners due to safetyand efficacy concerns, although homeopathic and cosmetic claims havebeen made since the 1950's. Mesotherapy was originally conceived inEurope as a method of utilizing cutaneous injections containing amixture of compounds for the treatment of local medical and cosmeticconditions. Although mesotherapy was traditionally employed for painrelief, its cosmetic applications, particularly fat and celluliteremoval, have recently received attention in the United States. One suchreported treatment for localized fat reduction, which was popularized inBrazil and uses injections of phosphatidylcholine, has been erroneouslyconsidered synonymous with mesotherapy. Despite its attraction as apurported “fat-dissolving” injection, the safety and efficacy of thesecosmetic treatments remain ambiguous to most patients and physicians.See, Rotunda, A. M. and M. Kolodney, Dermatologic Surgery 32: 465-480(2006) (“Mesotherapy and Phosphatidylcholine Injections: HistoricalClarification and Review”).

WO 2006/133160 (incorporated herein by reference in its entiretyincluding figures) describes methods for lipomodeling, e.g., reductionof a fat depot, by administering a neuropeptide Y receptor antagonist tothe site of the fat depot. Kolonin M. G. et al., Nat. Med. June10(6):625-32 (2004), describes fat selective pro-apoptotic peptideshaving potent fat cell killing effects. The described pro-apoptoticpeptides require access to the vasculature to kill.

Recently published literature reports that deoxycholic acid has fatremoving properties when injected into fatty deposits in vivo. See, WO2005/117900 and WO 2005/112942, as well as US2005/0261258;US2005/0267080; US2006/127468; and US20060154906, all incorporatedherein by reference in their entirety including figures). Deoxycholateinjected into fat tissue has two effects: 1) it kills fat cells via acytolytic mechanism; and 2) it causes skin tightening. Both of theseeffects are required to mediate the desired aesthetic corrections (i.e.,body contouring). Because deoxycholate injected into fat is rapidlyinactivated by exposure to protein and then rapidly returns to theintestinal contents, its effects are spatially contained. As a result ofthis attenuation effect that confers clinical safety, fat removaltherapies typically require 4-6 sessions. This localized fat removalwithout the need for surgery is beneficial not only for therapeutictreatment relating to pathological localized fat deposits (e.g.,dyslipidemias incident to medical intervention in the treatment of HIV),but also for cosmetic fat removal without the attendant risk inherent insurgery (e.g., liposuction). See, Rotunda et al., Dermatol. Surgery 30:1001-1008 (2004) (“Detergent effects of sodium deoxycholate are a majorfeature of an injectable phosphatidylcholine formulation used forlocalized fat dissolution”) and Rotunda et al., J. Am. Acad. Dermatol.(2005: 973-978) (“Lipomas treated with subcutaneous deoxycholateinjections”), both incorporated herein by reference.

Pharmaceutical grade bile acid preparations are commercially availableat relatively low cost. This low cost is due to the fact that the bileacids are obtained from animal carcasses, particularly large animalssuch as cows and sheep. Importantly, as with all medicaments from animalsources, there is concern that the animal-derived bile acid products maycontain animal pathogens and other harmful agents such as animal ormicrobial metabolites and toxins, including bacterial toxins such aspyrogens.

Such animal pathogens can include prions, which are thought to be a typeof infectious pathogenic protein that may cause prion diseases. Priondiseases are degenerative disorders of the nervous system. One suchdisease, “Mad cow” disease (thought to be a variant of Creutzfeldt-Jakobdisease (CJD)), is thought to be caused by a prion present in ediblebeef from diseased cows. Most cases are sporadic with unknown mode oftransmission; some cases are inherited; and a small number have beentransmitted by medical procedures. The spread of human prion diseasesthrough consumption of infected material has been implicatedhistorically in kuru and recently in variant CJD. Other animal priondiseases (scrapie of sheep, transmissible mink encephalopathy, chronicwasting disease of cervids, and bovine spongiform encephalopathy) allseem to be laterally transmitted by contact with infected animals or byconsumption of infected feed. Risk assessment and predictions of futureevents pertaining to prion diseases are difficult to ascertain becauseof the different modes of transmission, the unpredictable speciesbarriers, the variable distribution of infectivity in tissues, andstrain variations found in some diseases.

In general, animal products may be exposed to microbial organisms whichproduce pyrogens (fever-causing substances). Bacterial contaminants offood and/or pharmaceutical products are also a serious issue asevidenced by contamination of food stuffs by enterohemoragic E. coli.Products such as meats derived from cows as well as produce such asapples, spinach, and the like, have been implicated in suchcontamination. In such cases, it is the toxin produced by the bacteria(rather than the bacteria itself) that produces adverse effects inhumans. Such adverse effects include severe diarrhea, kidney failure andin the extreme situations, death. Bacterial endotoxins, a type ofpyrogen, must be substantially excluded from all pharmaceuticalcompositions.

Animal products are generally purified by a process of elimination,i.e., rather than selecting the end-product from a mix, the end productis the material remaining after exclusion of impurities. And, inaddition to the potential animal moieties such as pathogens, anotherartifact of purification from animal sources is that the end-product isa mixture of one or more bile acids. For example, commercialpreparations of deoxycholic acid contain some chenodoxycholic acid, aswell as cholic acid, which is a precursor to both deoxycholic acid andchenodeoxycholic acid in mammalian bile acid synthesis. Because theexact proportion of deoxy/cheno/cholic is not preselected, this mayresult in lot-to-lot variation when contemplating manufacturing largeamounts of bile acids. Such lot-to-lot variation can be problematic andmay engender additional steps in garnering regulatory approvals orquality control, particularly in efforts to produce a pharmaceuticalcomposition. Clearly, producers would desire lot-to-lot predictabilityin manufacturing bile acid pharmaceutical compositions.

Currently, the concerns regarding animal-derived products containinganimal pathogens and other harmful agents has been addressed by sourcingfrom isolated and inspected animals. For example, deoxycholic acid fromanimals in New Zealand are a source of bile acids for human use under USregulatory regimes, as long as the animals continue to remain isolatedand otherwise free of observable pathogens.

Implicitly, by the need for such governmentally controlled regulatoryregime is the recognition of an intrinsic risk of transmission of animalpathogens when animal-derived medicaments are injected. Where non-animalmedicament alternatives become available, the governmental regulatoryregime is no longer needed. An example of such alternative (non-animalmedicament replacing animal-derived medicament) and associatedadvantages is insulin for human use. The manufacture of beef insulin inthe United States was discontinued in 1998, and pork insulin for humanuse was discontinued in January of 2006. Although animal insulin can beobtained from herds not known to have had exposure to BSE-causing orother pathogenic agents, the manufacturing facilities or processes canexpose the animal ingredients to animals which have had exposure to thepathogens. The risk of transmission of pathogenic agents to humans canbe eliminated with the use of insulin that is manufactured recombinantlyor synthetically. For consumers, the insulin situation is instructive:where synthetic material is freely available, the risk of transmissionof animal pathogens is in theory eliminated. For producers, the abilityto produce a pure chemical entity that is substantially free of materialof animal pathogens is advantageous for safety, quality, and regulatorypurposes. Further, a synthetic process typically provides for a morereproducible product than that derived from biological sources.

Presently, because of the relative abundance of animal carcass-derivedbile acids, the industry has not taken steps to either fully chemicallysynthesize bile acids, or prepare bile acids using phytosterol ormicrobial starting materials. And although bile acid derivatives havebeen synthesized, this work again primarily involved animal-derived bileacids as starting materials for steroid chemistry, due to the low costand ready availability of animal materials. Despite historically activeefforts in phytosterol research, there are no readily-commerciallyavailable phytosterol-derived bile acid pharmaceutical gradecompositions. See, e.g., Mukhopadhyay, S. and U. Maitra., CurrentScience 87: 1666-1683, 1670 (2004) (Noting that the total synthesis ofany bile acid had not been performed subject to a 1981 reference,Kametani et al. J. Am. Chem. Soc. 103: 2890 (1981)(“First TotalSynthesis of (+)-Chenodeoxycholic Acid”). Microbial, such asbacterially-produced bile acids, have been used in situ as bacterialproducts, e.g., for marine oil spill clean-up. See, Maneerat et al.,Appl. Microbiol. Biotechnol. 76: 679-683 (2004) (“Bile acids are newproducts of a mariene bacterium, Myroides sp. Strain SM1”).

In order to realize the full potential of deoxycholic acid for theremoval of fat, it is imperative that the concerns over the use ofanimal derived products be further addressed. Clearly, there is a needfor suitable quantities of efficacious bile acids and relatedcompositions, such as the deoxycholic acids, that are known from theoutset to be free from moieties of animal origin (or pathogenic moietiescapable of acting in an animal, particularly a mammal, and for humanuse, having a deleterious effect on a human), and other harmful agentssuch as animal or microbial metabolites, toxins, including bacterialtoxins, such as pyrogens, for use as medicaments in humans. The presentinvention addresses this concern by providing synthetically preparedbile acid compositions free of the potential risk of animal pathogensand other harmful agents. The disclosed bile acid compositions can beused in adipolytic therapy and will serve to further advance researchand developmental efforts in the area of localized fat removal.

SUMMARY OF THE INVENTION

Adequate quantities of suitable bile acid as a defined pharmaceuticalcomposition is herein provided, as well as methods for synthesisthereof. Bile acid compositions and methods so provided are not isolatedfrom mammalian or microbial organisms that naturally produce the bileacids. In one aspect, particular deoxycholic acid pharmaceuticalcompositions which are free of all moieties of animal origin and ofmammalian and/or bacterial pyrogens, and related methods for productionand use are provided. In another aspect, adequate quantities of suitabledeoxycholic acids as defined pharmaceutical compositions are providedwhich can be used as an injectable pharmaceutical composition forlocalized fat removal, along with related compositions, methods formanufacture and methods of use. The defined deoxycholate injectates ofthe present invention may be combined with a molecule that causes fat todie by an orthogonal mechanism, e.g., NPY antagonists and/or fatselective pro-apoptotic peptides, to provide agents to be used to createa more potent means to mediate body contouring in fewer therapeuticsessions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing representing the structure of bile acids, includingthe numbering system for the carbons of the bile acid skeleton.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference into the presentdisclosure to more fully describe the state of the art to which thisinvention pertains.

As used herein, certain terms have the following defined meanings.

The term “pathogen” refers to a specific causative agent of a disease.

The term “animal origin” refers to originating from any of a kingdom(Animalia) of living things including many-celled organisms and singlecelled organisms.

The term “mammalian origin” refers to originating from any mammalianorganism. The term “mammalian organism” refers to a class (Mammalia) ofwarm-blooded higher vertebrates (as placentals, marsupials, ormonotremes) that nourish their young with milk secreted by mammaryglands, have the skin usually more or less covered with hair, andinclude humans.

The term “microbial origin” refers to originating from any microbialorganism. The term “microbial organism” refers to a domain (Bacteria) ofprokaryotic round, spiral, or rod-shaped single-celled microorganismsthat may lack cell walls or are gram-positive or gram-negative if theyhave cell walls, that are often aggregated into colonies or motile bymeans of flagella, that typically live in soil, water, organic matter,or the bodies of plants and animals, that are usually autotrophic,saprophytic, or parasitic in nutrition, and that are noted for theirbiochemical effects and pathogenicity.

The term “lower alkyl” refers to monovalent saturated aliphatichydrocarbyl groups having from 1 to 10 carbon atoms and preferably 1 to6 carbon atoms. This term includes, by way of example, linear andbranched hydrocarbyl groups such as methyl (CH₃—), ethyl (CH₃CH₂—),n-propyl (CH₃CH₂CH₂—), isopropyl ((CH₃)₂CH—), n-butyl (CH₃CH₂CH₂CH₂—),isobutyl ((CH₃)₂CHCH₂—), sec-butyl ((CH₃)(CH₃CH₂)CH—), t-butyl((CH₃)₃C—), n-pentyl (CH₃CH₂CH₂CH₂CH₂—), and neopentyl ((CH₃)₃CCH₂—).

The term “ethane dithiol or dithiane precursor” refers to a reagentthat, with reaction with a carbonyl group, will form an ethane dithiolor dithiane group.

The term “oxidizing agent” refers to a reagent which can acceptelectrons in an oxidation-reduction reaction. In this way, halogen oroxygen can be added to a molecule or hydrogen can be removed from amolecule.

The term “desulfurization reagent” refers to a reagent which can reactwith a sulfide. In one aspect, a desulfurization reagent can react witha sulfide containing molecule to remove the sulfide group from themolecule.

The term “reducing agent” refers to a reagent which can donate electronsin an oxidation-reduction reaction. In this way, halogen or oxygen canbe removed from a molecule or hydrogen can be added to a molecule.

The term “electrophilic acetyl group” refers to an acetyl group as anelectrophile, a group which is attracted to electrons and tends toaccept electrons.

The term “acetylating reagent” refers to a reagent in which can add anacetyl group to a molecule.

The term “acid” refers to a proton donor.

The term “hydrogenation reagent” refers to a reagent that can donatehydrogen to a molecule.

The term “dehydration reagent” refers to a reagent that can react withwater. In one aspect, a dehydration reagent can react with water that isremoved from a molecule.

In various aspects described herein, the present invention providescompositions (and useful intermediates) for pharmaceutical use, methodsof synthesis thereof, and methods of use of the present pharmaceuticalcompositions.

Importantly, the present bile acid compositions are free of risksinherent in material obtained from animal starting materials, andtherefore do not require the detailed inspections and regulations ofanimal-derived materials. In one aspect, this invention is thus directedto bile acid pharmaceutical compositions free of material of animalorigin, such as mammalian pathogens, as well as being substantially freeof toxins of bacterial origin, such as pyrogens. The present bile acidpharmaceutical compositions are optionally in salt form, and, furtheroptionally contain a pharmaceutically acceptable diluent, excipient orcarrier. Cations for salt preparation may be selected from the groupconsisting of sodium (Na⁺), potassium (K⁺), lithium (Li⁺), magnesium(Mg²⁺), calcium (Ca²⁺), barium (Ba²⁺), strontium (Sr²⁺), and ammonium(NH₄ ⁺). Salts may also be prepared from an alkali metal or an alkalineearth metal. An alkali metal may be selected from among sodium (Na⁺),potassium (K⁺), and lithium (Li⁺). An alkaline earth metal may beselected from the group consisting of magnesium (Mg²⁺), calcium (Ca²⁺),barium (Ba²⁺), and strontium (Sr²⁺). Preferably for use as apharmaceutical composition for localized removal of fat, the bile saltis sodium deoxycholate.

Prodrugs of the compounds of the embodiments are also contemplated. Aprodrug is an active or inactive compound that is modified chemicallythrough in vivo physiological action, such as hydrolysis, metabolism andthe like, into a compound of the embodiments following administration ofthe prodrug to a patient. For example, one may prepare an ester of thepresent deoxycholic acid or derivatives thereof, so that the release ofthe deoxycholic acid or derivatives thereof is triggered by thedisruption of the cell membrane, and release of esterase. With therelease of esterase, the ester protecting group is cleaved so that thedeoxycholic acid active form or derivatives thereof is present at thedesired location in situ. For a general discussion of prodrugs involvingesters see Svensson and Tunek Drug Metabolism Reviews 165 (1988) andBundgaard Design of Prodrugs, Elsevier (1985), herein entirelyincorporated by reference.

In general, the compounds of preferred embodiments will be administeredin a therapeutically effective amount by any of the accepted modes ofadministration for agents that serve similar utilities. The actualamount of the compound of preferred embodiments, i.e., the activeingredient, will depend upon numerous factors such as the severity ofthe disease to be treated, the age and relative health of the subject,the potency of the compound used, the route and form of administration,and other factors. The drug can be administered more than once a day,preferably once or twice a day. All of these factors are within theskill of the attending clinician.

The compositions can be comprised of a disclosed compound in combinationwith at least one pharmaceutically acceptable excipient. Acceptableexcipients are non-toxic, aid administration, and do not adverselyaffect the therapeutic benefit of the disclosed compound. Such excipientmay be any solid, liquid, semi-solid or, in the case of an aerosolcomposition, gaseous excipient that is generally available to one ofskill in the art.

Solid pharmaceutical excipients include starch, cellulose, talc,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, magnesium stearate, sodium stearate, glycerol monostearate, sodiumchloride, dried skim milk and the like. Liquid and semisolid excipientsmay be selected from glycerol, propylene glycol, water, ethanol andvarious oils, including those of petroleum, vegetable or syntheticorigin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.Preferred liquid carriers, particularly for injectable solutions,include water, saline, aqueous dextrose, and glycols.

The amount of the compound in a formulation can vary within the fullrange employed by those skilled in the art. The present compositions invarious aspects as described herein may be prepared wherein thedeoxycholic acid moiety is in the range of about 0.5%-10% on a weightper aqueous volume basis, or, on a w/w basis assuming the density ofwater (i.e., a 1:1 correspondence between weight and volume). In anotheraspect, the present embodiments relate to the presently describedpharmaceutical compositions in concentrations up to saturation point ofthe diluent. One may select the degree of thixotropic viscosity based onconditions such as concentration and pH. See, e.g., Mukhopadhyay, S. andU. Maitra, Current Science 87: 1666-1683 (2004) at 1680.

Of particular note is the potential for local irritation upon injectionof a bile acid composition of the present embodiments, and thus it maybe desirable to administer, simultaneously or in seriatim, a localanesthetic. For example, lidocaine is frequently used in humans, and maybe administered either as a co-formulation (in the same container andinjected at the same time) or co-injection (injected from a differentcontainer). Anesthetics such as lidocaine may be administered viatopical preparation, such as a patch or ointment.

For deeper tissue, anesthetics may be more deeply injected into thesubject tissue or administered systemically (e.g., general anesthesia,epidural or other known methods).

The bile acid(s) or bile salt(s) in a solution of the invention can beat a concentration of about 0.001 to 10, 0.01 to 5, or 0.1 to 2% w/w,w/v, or v/v. Preferably, the bile acid(s) or bile salt(s) in the abovesolution can be at a concentration of about 0.1-5% w/w or morepreferably about 1% w/w. In some embodiments, the fat dissolvingsolution comprises up to 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.05, 0.02,or 0.01 grams of the one or more detergents, bile acids and/or bilesalts, deoxycholic acid or salts thereof or sodium deoxycholate.

In preferred embodiments, the solutions herein include no lipids,phospholipids, or phosphatidylcholine. In some embodiments, thesolutions herein include up to 5% w/w, w/v, or v/v lipids,phospholipids, or phosphatidylcholine.

In some embodiments, the above solution can further comprise a secondtherapeutic agent selected from the group consisting of: anti-microbialagents, vasoconstrictors, anti-thrombotic agents, anti-coagulationagents, suds-depressants, anti-inflammatory agents, analgesics,dispersion agents, anti-dispersion agents, penetration enhancers,steroids, tranquilizers, muscle relaxants, and anti-diarrhea agents. Insome embodiments, a solution is in a container that contains up to 500mL of solution. Such container can be a syringe or syringe-loadablecontainer.

In some embodiments, compositions and methods further comprise amolecule known to cause fat to die by an orthogonal mechanism. Suchmolecules include neuropeptide Y (NPY) antagonists including, but notlimited to, NPY receptor antagonists, such as BIBP-3226 (Amgen),BIBO-3304 (Boehringer Ingleheim), BMS-192548 and AR-H040922(Bristol-Myers Squibb), LY-357897 (Eli Lilly), 1229U91 and GW4380145(GlaxoSmithKline), JNJ-5207787 (Johnson & Johnson), Lu-AA-44608(Lundbeck), MK-0557 (Merck NPY), NGD-95-1 (Neurgogen), NLX-E201(Neurologix), CGP-71683 (Novartis), PD-160170 (Pfizer), SR-120819A,BIIE0246, and S.A.0204 (Sanofi Aventis), S-2367 (Shiongli),dihydropyridine and dihydropyridine derivatives that are NPY receptorantagonists, bicyclic compounds that are NPY receptor antagonists,carbazole NPY receptor antagonists, and tricyclic compounds that are NPYreceptor antagonists. See, e.g., WO 2006/133160 and U.S. Pat. No.6,313,128 (incorporated herein by reference in its entirety includingfigures). Also contemplated are fat selective pro-apoptotic peptidessuch as the CKGGRAKDC peptide that homes to white fat vasculature. See,Kolonin M. G. et al., Nat. Med. June 10(6):625-32 (2004).

In some embodiments, the administering step involves delivering thecompositions herein via a dermal patch, a pump, or subdermal depot. Insome embodiments, the administering step involves delivering thecompositions herein topically or subcutaneously. In specificembodiments, the administration step involves administering locally(e.g., subcutaneously or subdermally) to a region under eye, under chin,under arm, buttock, calf, back, thigh, or stomach of said subject. Theadministration can be made by a subcutaneous or transdermal injection.

In one aspect, the present invention relates to methods for reducing asubcutaneous fat deposit in a subject. Such methods comprise the step ofadministering locally to a subcutaneous fat deposit in the subject acomposition comprising: (i) a fat-dissolving effective amount of one ormore pharmacologically active detergents, or bile acid(s) and/or bilesalt(s), or deoxycholic acid or a salt thereof, or sodium deoxycholate;(ii) a pharmaceutical, veterinary, or cosmetic excipient; and (iii)optionally a lipid, wherein the ratio of the lipid and bile acid or bilesalt is up to 1% w/w and wherein the composition does not include lipaseor colipase. In some embodiments, the fat deposit is associated with acondition selected from the group consisting of obesity, fatredistribution syndrome, eyelid fat herniation, lipomas, Dercum'sdisease, lipodystrophy, buffalo hump lipodystrophy, dorsocervical fat,visceral adiposity, breast enlargement, hyperadiposity, diffused bodyfat around trunk and arms, and fat deposits associated with cellulite.In preferred embodiments, the above method does not include performingsurgery on said subject.

In one aspect, the present invention relates to methods for reducing theappearance of a skin condition in a skin region of a subject. Suchmethods comprise the step of: administering locally to said skin regiona composition comprising: (i) a skin-tightening effective amount of oneor more pharmacologically active detergents, or bile acid(s) and/or bilesalt(s), or deoxycholic acid or a salt thereof, or sodium deoxycholate,(ii) a pharmaceutical, veterinary, or cosmetic excipient, and (iii)optionally a lipid. In some embodiments, the administering step involvesdelivering the compositions herein via a subcutaneous or transdermalinjection. In some embodiments, the skin condition being treated orameliorated is selected from the group consisting of: loose skin, skinaging, irregularities of the skin, and wrinkles. In some embodiments,the region of skin being treated is under eye, under chin, under arm,buttock, cheek, brow, calf, back, thigh, ankle, or stomach.

In some embodiments, the compositions used for reducing the appearanceof a skin condition in a skin region are formulation into a skintightening solution. Such skin tightening solution can further comprisea second therapeutic agent selected from the group consisting of:anti-microbial agents, vasoconstrictors, anti-thrombotic agents,anti-coagulation agents, suds-depressants, anti-inflammatory agents,analgesics, dispersion agents, anti-dispersion agents, penetrationenhancers, steroids, tranquilizers, muscle relaxants, and anti-diarrheaagents.

In preferred embodiments, the detergent comprises a bile acid selectedfrom the group consisting of deoxycholic acid, cholic acid,chenodeoxycholic acid, 7-alpha-dehydroxylate chenodeoxycholic acid,lithocholic acid, ursodeoxycholic acid, dihydroxytaurin acid,trihydroxytaurine acid, and glycine conjugates of any of the above. Insome embodiments, the detergent comprises a bile salt that includes acation selected from the group consisting of sodium (Na⁺), potassium(K⁺), lithium (Li⁺), magnesium (Mg²⁺), calcium (Ca²⁺), barium (Ba²⁺),strontium (Sr²⁺), and ammonium (NH₄ ⁺). In some embodiments, thedetergent comprises a bile salt with a cation that is an alkali metal oran alkaline earth metal. Preferably, the alkali metal is sodium (Na⁺),potassium (K⁺), or lithium (Li⁺) and the alkaline earth metal ismagnesium (Mg²⁺), calcium (Ca²⁺), barium (Ba²⁺), or strontium (Sr²⁺).More preferably, the bile salt is sodium deoxycholate.

Sodium deoxycholate is a naturally produced bile salt that solubilizesdietary lipids in the digestive tract. It is produced in vivo via acomplex biosynthetic route utilizing cholesterol as the startingmaterial and involving both human and bacterial enzymes. The primaryfunction of deoxycholate is to assist in the digestive process bysolubilizing dietary lipids to facilitate absorption. In the body,deoxycholate biosynthesis begins with the enzymatic oxidation,isomerization, and reduction of cholesterol in the liver to form cholicacid, a bile acid structurally similar to its cholesterol parent (StryerL, Chapter 27: Biosynthesis of Membrane Lipids and Steroids, inBiochemistry, 1995, W. H. Freeman and Company: New York. p. 691-707). Inthe liver, cholic acid is then chemically linked to one of two aminoacids (taurine or glycine) to form the ‘conjugated’ cholic acids (i.e.,L-glycocholate and taurocholate). These conjugated cholic acids are thenstored in the gall bladder until food consumption. After foodconsumption, bile solution is released from the gall bladder into theintestine, where the conjugated cholic acid molecules are subject to twoadditional chemical modifications mediated by enzymes produced byintestinal microflora (Ridlon J. M., Kang D. J. and Hylemon P. B., Bilesalt biotransformation by human intestinal bacteria, J. Lipid Res.,2006, 47(2): p. 241-59). First, conjugated cholic acid is dehydroxylatedto form conjugated deoxycholate. Conjugated deoxycholate is thendeconjugated to form free deoxycholate, which participates, along withthe other bile acids, in the solubilization of dietary lipids. Becausedeoxycholate is downstream from cholic acid synthesis, cholic acid maybe an impurity present in natural sources of deoxycholate.

Deoxycholate is soluble to 333 mg/mL in water, sparingly soluble inalcohol, and is even less soluble in acetone and glacial acetic acid.Reversible formation of micelles may occur with sodium deoxycholateconcentrations above the critical micelle concentrations ofapproximately 2.4 mg/mL and neutral pH (Matsuoka K, M. Y., Micelleformation of sodium deoxycholate and sodium ursodeoxycholate (part 1),Biochim. Biophys. Acta., 2002, 1580(2-3): p. 189-99). At concentrationsabove the critical micelle concentration of 2.4 mg/mL, deoxycholate willform micelles and has the ability to solubilize cells, lipids, andproteins. At lower concentrations such as 0.4 mg/mL (comparable to thefasting state), deoxycholate is 98% bound to albumin (Roda A. et al.,Quantitative aspects of the interaction of bile acids with human serumalbumin, J. Lipid Res., 1982. 23(3): p. 490-5) in the presence of 26mg/mL of albumin (which is close to the serum physiologicalconcentration of 35-50 mg/mL).

The preferred embodiments are directed to deoxycholic acid (DCA) or apharmaceutically acceptable salt thereof and the related compositionsand methods, wherein deoxycholic acid (DCA) is:

wherein said compound is not isolated from a mammalian or microbialorganism naturally producing DCA.

Other preferred embodiments also are directed to stereoisomers of DCAand pharmaceutically acceptable salts thereof and to intermediates inthe synthesis of the DCA and its stereoisomers and salts and the relatedcompositions and methods.

Methods for complete chemical synthesis of bile acid pharmaceuticalcompositions, and useful intermediates, are now provided.

These following descriptions and examples provide an alternative to theextraction of DCA from mammalian or microbial organisms that naturallyproduce this compound. Synthetic routes 1-6 are contemplated for use inthe present invention to synthesize deoxycholic acid (DCA). Syntheticroute 1B and Examples 1-11 show the synthesis of DCA fromhydrocortisone.

1. Synthetic Route #1A from Adrenosterone, Via 9(11)-ene or 11,12-ene

Cortisone (Compound 1.1) of Scheme 1A (below) is widely available as afully synthetic material. It can be efficiently cleaved to form the C₁₇ketone compound using pyridinium chlorochromate (PCC). This cleavage toadrenosterone (Compound 1.2) can also be achieved using HIO₄ or sodiumbismuthate (NaBiO₃). The reaction that converts Compound 1.2 to Compound1.3 is a known chemical process. Conversion of Compound 1.3 intoCompound 1.4 involves monoketalization. Subsequent steps areregeneration of the 3-keto-4-ene, selective reduction of the 4,5-ene(H₂/Pt/DMF) to yield the C₅ β-configuration and selective reduction ofthe C₃ carbonyl group to the desired 3α-configuration to yield compound1.5. The addition of a protecting group in converting Compound 1.5 intoCompound 1.6 and subsequent reduction of the product yields the C₁₁ β-ol(axial configuration), i.e., compound 1.7, which is suitable forregioselective elimination to the key 9(11)-ene (i.e., conversion ofCompound 1.7 into Compound 1.8).

The synthetic scheme bifurcates here, in that Compound 1.7 can be usedas the starting material for conversion to either Compound 1.8 orCompound 1.9. The elimination reaction used to convert Compound 1.7 intoCompound 1.8 is regioselective because of the trans diaxial relationshipbetween the C₁₁ hydroxyl group and C₉ hydrogen atom. The alternativemode of elimination to yield the isomeric C₁₁-C₁₂ olefin of compound 1.9is likewise regioselective involving cis-thermal elimination (i.e.,conversion of Compound 1.7 to Compound 1.9).

Allylic oxidation of Compound 1.8 (via treatment with CrO₃ and 3,5dimethyl pyrazole) yields the enone-containing Compound 1.10. Peracidoxidation of Compound 1.9 proceeds stereoselectively from the alpha-faceof the steroid to yield the C₁₁₋₁₂ epoxide Compound 1.11 (see Scheme 1Aabove). These chemical transformations yield the two key precursors ofthe C₁₂ hydroxyl group functionality, namely, Compound 1.10 and Compound2.1 (Schemes 1A and 2).

One of the skill in the art will appreciate that the above cortisoneroute can be modified to begin instead with hydrocortisone, which hasthe same carbon skeleton and the same relative placement of oxygenatoms, with hydrocortisone differing from cortisone only in theoxidation state of the C-11 oxygen bearing carbon atom. Hydrocortisoneis commercially available and various synthesis of this compound areknown (Szczebara et. al. Nature Biotechnology Vol. 21, February 2003,143-149) including a total chemical synthesis (Woodward R. B. et. al.1952, J. Am. Chem. Soc. 74: 4223). Ketone 1.13 is synthesized startingfrom hydrocortisone 1.12 (Scheme 1B) via hydrogenolysis of theα,β-unsaturated double bond, followed by global ketone reduction usingsodium borohydride to allow for 1,2-diol cleavage using NaIO₄, thusforming the C₁₇ ketone on the D-ring of the steroidal ring system.Subsequent oxidation with pyridinium chlorochromate (PCC) yields 1.13.Treatment of 1.13 with K-SELECTRTDE® (potassiumtri-sec-butylborohydride) followed by acetylation with aceticanhydride/pyridine gives protected alcohol 1.15. Subsequent olefinationof 1.15 with a Wittig reagent provides alkene 1.16 that is then treatedwith methyl propiolate and ethyl aluminum dichloride to form diene 1.17.Following hydrogenation of both double bonds, ketone 1.18 is reduced andthe resulting alcohol intermediate is eliminated upon treatment withSOCl₂ in pyridine to give alkene 1.19. Allylic oxidation of alkene 1.19with CrO₃ and reduction of the double bond under hydrogenationconditions gives ketone 1.21. Removal of the acetate protecting groupand oxidation of the resulting alcohol gives diketone 1.22. Reduction of1.22 with LiAlH(O-^(t)Bu)₃ and hydrolysis of the methyl ester yieldsDCA.

Further transformations of Compounds 1.10 and 2.1 are shown in Scheme 2.First, Compound 1.10 is modified to contain a properly functionalized Cring system identical to that of DCA (Scheme 2). Stereoselectivereduction of the C₁₂ carbonyl group yields Compound 2.1 and catalytichydrogenation of the 9(11) double bond present in Compound 2.1 yieldsCompound 2.2.

Scheme 3 presents the transformation of epoxide-containing Compound 1.11to the analogous C₁₂ α-hydroxy steroid Compound 2.2 of Scheme 2.

As mentioned above in both of these routes common intermediate compound2.2 is formed.

The next step in the synthesis of DCA is the modification of the D-ringpresent in Compound 2.2 such that it contains the carboxylic side chainsubstituted D ring of DCA (Scheme 4 and Scheme 5).

First the C₁₇ ketal and the C₃ silyl ether groups of Compound 2.2 arehydrolyzed. Then the Wittig reaction is performed to yield Compound 4.2.Conversion of Compound 4.2 to Compound 5.1 is carried out via an enereaction. Subsequent catalytic reduction of Compound 5.1 and hydrolysisof the ester yields DCA (Scheme 5).

2. Synthetic Route #2 from Cortisone Via Adrenosterone (the i-Steroid,3,5-Cyclosterol Route)

Selective ketalization of adrenosterone (Compound 1.2, Scheme 6) at C₁₇,borohydride reduction, mesylation, and buffered hydrolysis yields thei-steroid (3,5-cyclosterol) containing Compound 6.1. Compound 6.1undergoes 9(11)-ene formation (conversion of Compound 6.1 to Compound6.2, Scheme 6) and allylic oxidation (conversion of Compound 6.2 toCompound 6.3, Scheme 6) followed by carbonyl group reduction to yieldCompound 6.4. Hydrolysis of the i-sterol and hydrogenation yieldsCompound 6.5, which can be converted to DCA by synthetic methodspresented above in Synthetic Route #1.

3. Synthetic Route #3 from Hecogenin

Hecogenin (Compound 7.1, Scheme 7) is a plant sterol found abundantly inMexican yams and other plants of the Agave species. The centraladvantage of hecogenin as a starting material for DCA synthesis is thatit possesses a C₁₂ oxygen functionality as is present in DCA.

The first step in the synthetic route starting from hecogenin is thestereoselective reduction of the C₁₂ carbonyl group in hecogenin(Compound 7.1) to the requisite C₁₂-α configuration (conversion ofCompound 7.1 to Compound 7.2). Then the 3β-ol, 5α-AB ring system isconverted to the 3 α-ol, 5β-AB (Conversion of Compound 7.1, to Compound7.2, to Compound 7.3) ring system (Scheme 7). The well-known Markerdegradation (Marker, R. E., Rohrmann, E., Sterols. LXIX OxidationProducts of Sarsasapogenin. Sarsasapogenoic Acid and Related Substances,J. Am. Chem. Soc., 1939. 61(8): p. 2072-2077) follows the conversion ofCompound 7.2 to Compound 7.3 to yield Compound 7.4. Installation of theD-ring side chain (Scheme 8) into Compound 8.2 is achieved via methodsshown in Schemes 4 and 5. The requisite C₁₇ ketone in Compound 8.2 isformed by ozonolysis of the enol acetate of Compound 8.1 (Scheme 8). DCAis then prepared from 8.2 in a similar manner as in Scheme 5 using theolefination and ene reaction sequence. Alternative routes starting fromstarting from hecogenin are shown in Schemes 9 and 10.

4. Synthetic Route #4 from Sapogenins

Sapogenins are derived from the hydrolysis of the saccharides anddisaccharides attached to the C₃ hydroxyl group of the saponins (i.e.,steroid glycosides). These are widely occurring plant products. Saponinoccurs in nature as a spiroketal structure as shown below. Also Compound10.3 can be formed from tigogenin, diosgenin, chlorogenin, smilageninand hecogenin (Compound 7.1). We believe that DCA could be synthesizedfrom each of these, namely, tigogenin, diosgenin, chlorogenin,smilagenin and hecogenin (Compound 7.1). (Y. Mazur, N. Danieli and FranzSondheimer J. Am. Chem. Soc.; 82, 5809, 1960).

Since we could synthesize DCA from hecogenin, we recognize that any ofthe above sapogenins could, likewise, serve as a starting material forDCA synthesis.

5. Synthetic Route #5 from Stigmasterol

Stigmasterol (Compound 11.1) is a widely available plant sterol. As astarting material for the DCA synthesis, it has an advantage in that itcontains a functionalized AB ring system and a readily cleavable sidechain moiety. It has the disadvantage in that it lacks the requiredfunctionality in the C ring essential for DCA synthesis.

In this synthetic route, the stigmasterol (Compound 11.1) AB ring isprotected by i-steroid formation followed by ozonolysis to yield aside-chain at C₁₇ installed and reduced to the C₂₄-ol as a masked formof the carboxyl group (Scheme 11). The subsequent steps generate anallylic position at C₁₂ (Scheme 12). The B-ring diene formation andmercuric acetate oxidation are known processes and catalytic reductionof the B ring system yields an intermediate common with previous routesdescribed above. However, in contrast to other routes, the side chain isalready present. Allylic oxidation (conversion of Compound 12.4 toCompound 1.20) and stereoselective reduction (conversion of Compound1.20 to Compound 1.21) followed by previously discussed steps, yields aproduct which is converted to DCA.

A variation of the stigmasterol route uses the Diels-Alder protection ofthe B-ring diene. This is advantageous because it isolates the 9(11)double bond to prevent possible interference during the allylicoxidation steps (Scheme 13).

6. Synthetic Route #6 from Ergosterol

Ergosterol (Compound 14.1) is a readily available starting material andcan be used to prepare DCA by adaptation of the procedures set forth inthis application. Allylic oxidation offers a facile route to C₁₂ oxygenfunctionality (Scheme 14). This route has the advantage of starting withthe ring B diene. It is convergent with the stigmasterol route.

Another embodiment provides for a method for removal of fat depositsfrom selected locations in a mammal comprising administering to themammal in need thereof a therapeutically effective amount of a compoundthat is DCA or pharmaceutically acceptable a salt thereof,

wherein said compound is not isolated from a mammalian or microbialorganism naturally producing DCA.

Another embodiment provides for a method of emulsifying fat in a mammalcomprising administering to the mammal in need thereof a therapeuticallyeffective amount of a compound that is DCA or pharmaceuticallyacceptable a salt thereof,

wherein said compound is not isolated from a mammalian or microbialorganism naturally producing DCA.

Another embodiment provides for a method of solubilizingphosphatidylcholine comprising mixing phosphatidylcholine and effectiveamount of a compound that is DCA or pharmaceutically acceptable a saltthereof,

wherein said compound is not isolated from a mammalian or microbialorganism naturally producing DCA.

Another aspect of the invention relates to mixing adipo-ablative bileacids, such as, deoxycholic acid (DCA) with agents that kill fat cells.In one aspect, this invention contemplates a means to enhance theaesthetic effects of deoxycholate injections by mixing into thedeoxycholate injectate a molecule that causes fat to die by anorthogonal mechanism. Examples of such candidate molecules include, butare not limited to, neuropeptide Y (NPY) antagonists and fat selectivepro-apoptotic peptides. Since both fat cell killing and skin tighteningmay be required to mediate the desired effects, the effects of an agentwith fat killing ability and potent skin tightening effects (such asdeoxycholate) can be enhanced via the addition of a molecule with potentfat cell killing effects. Additionally, molecules that require access tothe vasculature to kill (such as certain pro-apoptotic peptides thatbind to proteins expressed on the luminal side of capillaries) can gainaccess to these proteins because deoxycholate may cause vascularleakage. Thus, such agents can be synergistic with deoxycholatepotentially creating a more potent means to mediate body contouring infewer therapeutic sessions.

Examples of NPY antagonists include, but are not limited to, NPYreceptor antagonists, such as BIBP-3226 (Amgen), BIBO-3304 (BoehringerIngleheim), BMS-192548 and AR-H040922 (Bristol-Myers Squibb), LY-357897(Eli Lilly), 1229U91 and GW4380145 (GlaxoSmithKline), JNJ-5207787(Johnson & Johnson), Lu-AA-44608 (Lundbeck), MK-0557 (Merck NPY),NGD-95-1 (Neurgogen), NLX-E201 (Neurologix), CGP-71683 (Novartis),PD-160170 (Pfizer), SR-120819A, BIIE0246, and S.A.0204 (Sanofi Aventis),S-2367 (Shiongli), dihydropyridine and dihydropyridine derivatives thatare NPY receptor antagonists, bicyclic compounds that are NPY receptorantagonists, carbazole NPY receptor antagonists, and tricyclic compoundsthat are NPY receptor antagonists. See, e.g., WO 2006/133160 and U.S.Pat. No. 6,313,128 (incorporated herein by reference in its entiretyincluding figures).

Exemplary fat selective pro-apoptotic peptides includes, but is notlimited to, CKGGRAKDC peptide that homes to white fat vasculature. See,Kolonin M. G. et al., Nat. Med. June 10(6):625-32 (2004).

The compounds of preferred embodiments can be prepared from readilyavailable starting materials using the following general methods andprocedures. It will be appreciated that where typical or preferredprocess conditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. Suitableprotecting groups for various functional groups as well as suitableconditions for protecting and deprotecting particular functional groupsare well known in the art. For example, numerous protecting groups aredescribed in T. W. Greene and G. M. Wuts, Protecting Groups in OrganicSynthesis, Third Edition, Wiley, New York, 1999, and references citedtherein.

The starting materials and reagents for the reactions described hereinare generally known compounds or can be prepared by known procedures orobvious modifications thereof. For example, many of the startingmaterials and reagents are available from commercial suppliers such asAldrich Chemical Co. (Milwaukee, Wis., USA), Bachem (Torrance, Calif.,USA), Emka-Chem or Sigma (St. Louis, Mo., USA). Others may be preparedby procedures, or obvious modifications thereof, described in standardreference texts such as Fieser and Fieser's Reagents for OrganicSynthesis, Volumes 1-15 (John Wiley and Sons, 1991), Rodd's Chemistry ofCarbon Compounds, Volumes 1-5 and Supplementals (Elsevier SciencePublishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley and Sons,1991), March's Advanced Organic Chemistry, (John Wiley and Sons, 4^(th)Edition), and Larock's Comprehensive Organic Transformations (VCHPublishers Inc., 1989).

The various starting materials, intermediates, and compounds of thepreferred embodiments may be isolated and purified where appropriateusing conventional techniques such as precipitation, filtration,crystallization, evaporation, distillation, and chromatography.Characterization of these compounds may be performed using conventionalmethods such as by melting point, mass spectrum, nuclear magneticresonance, and various other spectroscopic analyses.

EXAMPLES

The various starting materials, intermediates, and compounds of thepreferred embodiments may be isolated and purified where appropriateusing conventional techniques such as precipitation, filtration,crystallization, evaporation, distillation, and chromatography.Characterization of these compounds may be performed using conventionalmethods such as by melting point, mass spectrum, nuclear magneticresonance, and various other spectroscopic analyses.

Exemplary embodiments of steps for performing the synthesis of productsin Synthetic Route #1, Scheme 1B is described in greater detail infra.Table 1 describes abbreviations used to express variouscompounds/moieties/apparatus/procedure/property in the exemplaryreaction schemes and synthetic routes described in the followingexamples and throughout the specification.

TABLE 1 AcOH Acetic acid CAN Acetonitrile Ac₂O Acetic anhydride AcClAcetyl chloride NH₄Cl Ammonium chloride CHCl₃ Chloroform CrO₃ Chromiumtrioxide DCA Deoxycholic acid DCM (CH₂Cl₂) Dichloromethane DMFN,N-Dimethylformamide DMSO Dimethyl sulfoxide EtOAc Ethyl acetateEtAlCl₂ Ethyl aluminum dichloride Hz Hertz HPLC High pressure liquidchromatography HCl Hydrochloric acid LAH Lithium aluminum hydride LiOHLithium hydroxide MgSO₄ Magnesium sulfate MHz Megahertz MeOH Methanolmmol millimole mL milliliter mol mole Obs Observed HClO₄ Perchloric acidPtO₂ Platinum oxide KBr Potassium bromide K—O^(t)Bu Potassiumtert-butoxide PCC Pyridinium chlorochromate Rep Reported NaOH Sodiumhydroxide THF Tetrahydrofuran SOCl₂ Thionyl chloride TEA TriethylamineTLC Thin layer chromatography Wt Weight

General:

Manipulations of oxygen- and moisture-sensitive materials are conductedwith two-necked flame dried flasks under an argon atmosphere. Columnchromatography is performed using SE-Make silica gel (60-120 Mesh),Spectrochem silica gel (230-400 Mesh) or aluminium oxide 90-neutral.(SD-Fine Chem. Ltd., India). Analytical thin layer chromatography (TLC)was performed on Merck Kieselgel 60 F₂₅₄ (0.25 mm) plates (Merck & Co.,Whitehouse Station, N.J.). Visualization of spots was detected either byUV light (254 nm) lamp or by charring with a solution of sulfuric acid(5%) and p-anisaldehyde (3%) in ethanol.

Apparatus:

Analysis of the compounds and products of the reaction schemes andsynthetic routes described herein may be performed on the apparatus andequipment described infra.

Nuclear Magnetic Resonance (NMR)

Proton and carbon nuclear magnetic resonance spectra (¹H NMR and ¹³CNMR) are recorded on a Varian Mercury-Gemini 200 (¹H NMR, 200 MHz; ¹³CNMR, 50 MHz) or a Varian Mercury-Inova 500 (¹H NMR, 500 MHz; ¹³C NMR,125 MHz) (Varian, Inc., Palo Alto, Calif.) spectrometer with solventresonances as the internal standards (¹H NMR, CHCl₃ at 7.26 ppm or DMSOat 2.5 ppm and DMSO-H₂O at 3.33 ppm; ¹³C NMR, CDCl₃ at 77.0 ppm or DMSOat 39.5 ppm). ¹H NMR data are reported as follows: chemical shift (δ,ppm), multiplicity (s=singlet, d=doublet, t=triplet, q=quartet,br=broad, m=multiplet), coupling constants (Hz), and integration.

Infrared Spectroscopy

Infrared spectra (FT-IR) are run on a JASCO-460⁺ model (Jasco, Inc.,Easton, Md.). Mass spectra are obtained with a Perkin Elmer, API-2000spectrometer (Perkin Elmer, Inc., Waltham, Mass.) using ES⁺ mode.

Melting Point

Melting points were determined using a LAB-INDIA melting point measuringapparatus (Labindia Instruments Pvt. Ltd., India) and are uncorrected.

High Pressure Liquid Chromatography

HPLC chromatograms were recorded using a SHIMADZU-2010 model with a PDAdetector (Shimadza Corp., Japan).

Optical Activity

Specific optical rotations ([α]_(D)) are determined employing aJASCO-1020 at 589 nm (Jasco, Inc., Easton, Md.) and are uncorrected.

Chemicals:

Unless otherwise noted, commercially available reagents are used withoutpurification. Diethyl ether and THF are distilled fromsodium/benzophenone ketyl. Anhydrous DMF, DCM, pentane and hexane areobtained by distillation from CaH₂.

Example 1: Preparation of Androstane-3,11,17-trione (1.13)

10% of Pd/C (2.5 g, 5 wt %) is added to a solution of hydrocortisone(Compound 1.12) (50.0 g, 138.12 mmol) in DMF (250 mL). The resultingslurry is hydrogenated in a Parr apparatus (50 psi) for 12 h. Uponcomplete disappearance of starting material, as evidenced by TLC, thecrude reaction mixture is filtered through a small plug of Celite, andthe solvent is removed under vacuum. Crude product (48.0 g) is obtainedas a colorless solid.

NaBH₄ (2.1 g, 55.3 mmol) is added to a solution of the above crudeproduct (48.0 g, 131.86 mmol) in EtOH (500 mL) and CH₂Cl₂ (500 mL).After 1 hr, acetone (50 mL) and water (150 mL) are added, followed byNaIO₄ (70.5 g, 329.6 mmol). The mixture is stirred at room temperatureovernight.

Distilled water (500 mL) is added and the mixture is extracted withethyl acetate (3×250 mL). The ethyl acetate layer is flushed through asilica-gel plug and the solvent is evaporated to yield 38 g as acolorless solid. The crude product is oxidized further withoutpurification.

PCC (40.4 g, 187.5 mmol) is added to a solution of the above crudeproduct in CH₂Cl₂ (400 mL) in 3 equal portions over 30 minutes. Theresulting reaction mixture is stirred at room temperature for about 3-4h. Upon completion of the reaction, as monitored by TLC, the crudereaction mixture is filtered sequentially through pads of Celite andsilica gel and the crude material is purified by column chromatography[59 (W)×700 (L) mm, 60-120 Mesh silica, 150 g], eluting with ethylacetate/hexane (3:10) [50 mL fractions, 10 mL/min elution, monitored byTLC with p-anisaldehyde charring; R_(f) for Compound 1.13=0.37 and R_(f)for Compound 1.12=0.05 in EtOAc/Hexane (1:1)] to provide thediastereomeric Compound 1.13 (33.0 g, 79% yield) as a colorless solid.

The obtained crude material was purified by preparative HPLC using aPhenomenex Lunov C18 column (250×30.0 mm, 10μ) and isocratic elutionwith CH₃CN:H₂O (12:13) with a 25 mL/min flow rate in 15 mL fractions.The preparative HPLC is only used for purification, but not foranalysis. Table 2 describes the measured properties of the product.

TABLE 2 ¹H NMR δ = 2.76 (dt, J = 4.0, 15.0 Hz, 1H), 2.62-2.35 (m, 5H),2.33-2.24 (m, (500 MHz, CDCl₃) 1H), 2.23-2.05 (m, 4H), 2.02-1.88 (m,3H), 1.81 (bd, J = 14.0 Hz, 2H), 1.72-1.61 (m, 1H), 1.57-1.48 (m, 1H),1.47-1.32 (m, 2H), 1.26 (s, 3H), 0.86 (s, 3H) ¹³C NMR δ = 216.9, 211.8,208.4, 52.3, 50.3, 50.2, 50.0, 44.5, 41.9, 37.1, (125 MHz, CDCl₃) 36.0,35.9, 35.8, 34.3, 25.6, 25.0, 22.2, 21.3, 14.5 Mass (m/z) 303.2 [M⁺ +1], 320.1 [M⁺ + 18] IR (KBr) 3443, 2916, 1729, 1705, 1466, 1379, 1044cm⁻¹ m.p. 128.9-131° C. (from CH₂Cl₂/Hexane) (observed); 128-131° C.(Rep. E. Caspi J. Org. Chem. 1959, 24, 669) [α]_(D) +139 (c = 1 inCHCl₃). HPLC purity 98.6%, ret. time = 16.61, (Hypersil BDS C18; 250 ×4.6 mm, 5u), ACN: 5 mM TEA pH-2.5 with HClO₄ (Gradient), absorbance at205 nm

Example 2: 3β-Hydroxy-androstane-11,17-dione (1.14)

K-SELECTRIDE® (potassium tri-sec-butylborohydride, 98.39 mL, 98.01 mmol,1M solution in THF) is added to a solution of Compound 1.13 (33.0 g,109.27 mmol) in THF (330 mL) over 15 minutes under an inert atmosphereat −78° C. and is stirred for about 3-4 h at −78° C. The reactionmixture is quenched with aqueous NaOH solution (2M, 70 mL). The crudereaction mixture is diluted with ethyl acetate (500 mL) and the organiclayer is washed with water (3×75 mL), saturated brine solution (100 mL)and dried over MgSO₄ (75 g). The solvent is removed under vacuum toafford 33 g of crude material. The crude product is subjected toacetylation without purification.

Purification of Crude Material

The crude material is purified by column chromatography [29 (W)×600 (L)mm, 230-400 Mesh silica, 200 g], eluting with ethyl acetate/hexane (1:4)[25 mL fractions, 5 mL/min elution, monitored by TLC with p-anisaldehydecharring; R_(f) for Compound 1.14=0.3 and R_(f) for Compound 1.13=0.37in EtOAc/Hexane (1:1)] to afford Compound 1.14. Table 3 describes themeasured properties of the product.

TABLE 3 ¹H NMR δ = 4.08 (s, 1H), 2.53 (q, J = 9.0 Hz, 1H), 2.42 (d, 7 =13.0 Hz, (500 MHz, CDCl₃) 1H), 2.34-2.21 (m, 3H), 2.11-2.04 (m, 1H),1.98-1.91 (m, 3H), 1.88-1.59 (m, 6H), 1.57-1.26 (m, 6H), 1.21 (s, 3H),0.82 (s, 3H) ¹³C NMR δ = 217.4, 209.1, 66.3, 51.6, 50.6, 50.5, 37.1,36.2, 35.9, 34.8, 33.5, (125 MHz, CDCl₃) 28.8, 28.5, 25.7, 25.6, 23.6,21.5, 14.5 Mass (m/z) 305.0 [M⁺ + 1], 322.0 [M⁺ + 18] IR (KBr) 3519,2928, 1735, 1697, 1454, 1379 cm⁻¹ m.p. 176.6-180.5° C. [α]_(D) +125 (c =1 in CHCl₃)

Example 3: 3β-Hydroxyandrostane-11,17-dione acetate (1.15)

Acetic anhydride (16.6 g, 162.8 mmol) is added to a solution of Compound1.14 (33.0 g, 108.55 mmol) in pyridine (150 mL) at 0° C. under an inertatmosphere. The resulting reaction mixture is stirred overnight atambient temperature. Upon completion of the reaction, as evidenced byTLC, pyridine and remaining acetic anhydride are removed under vacuum.The crude residue is diluted with ethyl acetate (500 mL) and washed withwater (3×150 mL), saturated brine solution (100 mL) and dried over MgSO₄(75 g). The solvent is evaporated under vacuum and the crude material ispurified by column chromatography [59 (W)×800 (L) mm, 60-120 Meshsilica, 150 g], eluting with ethyl acetate/hexane (1:10) [25 mLfractions, 10 mL/min elution, monitored by TLC with p-anisaldehydecharring; R_(f) for Compound 1.15=0.38 and R_(f) for Compound 1.14=0.1in EtOAc/Hexane (3:7)] to afford Compound 1.15 (19.0 g, 66.4% yield) asa colorless solid. Table 4 describes the measured properties of theproduct.

TABLE 4 ¹H NMR δ = 5.03 (s, 1H), 2.53 (dd, J = 9.5, 19.0 Hz, 1H), 2.42(d, J = 10.0 (500 MHz, CDCl₃): Hz, 1H), 2.36-2.31 (m, 3H), 2.25 (dd, J =9.5, 19.0 Hz, 1H), 2.10- 2.06 (m, 1H), 2.04 (s, 3H), 1.96-1.91 (m, 3H),1.81-1.69 (m, 2H), 1.63-1.57 (m, 3H), 1.50 (dd, J = 3.0, 14.5 Hz, 1H),1.36 (d, J = 9.5 Hz, 3H), 1.27-1.22 (m, 1H), 1.20 (s, 3H), 0.82 (s, 3H)¹³C NMR δ = 217.2, 208.9, 170.4, 69.7, 51.5, 50.5, 50.4, 37.9, 36.1,35.9, (125 MHz, CDCl₃) 34.5, 30.6, 29.6, 29.5, 25.5, 25.4, 25.3, 23.4,21.4, 21.3, 14.5 Mass (m/z) 347.1 [M⁺ + 1], 364.1 [M⁺ + 18] IR (KBr)3455, 2927, 1737.6, 1720.2, 1707.7, 1259, 1244 cm⁻¹ m.p. 156-158° C.[α]_(D) 116 (c = 1 in CHCl₃)

Example 4: (Z)-3β-Hydroxy-5β-preg-17(20)-ene-11-one acetate (1.16)

Potassium tert-butoxide (159.28 mL, 159.2 mmol, 1M solution in THF) isadded to a solution of ethyltriphenylphosphonium bromide (61.16 g, 164.8mmol) in THF (150 mL) is added drop wise over 1 h under an inertatmosphere at −5° C. The resulting dark pink colored reaction mixture iswarmed to 10-15° C. and stirred for an additional 1 h at the sametemperature. A solution of Compound 55 (19.0 g, 54.9 mmol) in THF (50mL) is introduced slowly to the above Wittig ylide suspension at −5° C.The solution is stirred for an additional 10-20 minutes and the reactionmixture is allowed to warm to ambient temperature slowly. Stirring iscontinued for about 3-4 h. Upon complete disappearance of startingmaterial, as evidenced by TLC, the reaction mixture is quenched withsaturated aqueous NH₄Cl solution (75 mL). The aqueous layer is extractedwith EtOAc (2×150 mL) and the combined organic extracts are washed withsaturated brine solution (100 mL) and dried over MgSO₄ (75 g). Thesolvent is removed under vacuum and the crude material is purified bycolumn chromatography [49 (W)×600 (L) mm, 60-120 Mesh silica, 300 g]eluting with ethyl acetate/hexane (1:20) [25 mL fractions, 10 mL/minelution, monitored by TLC with p-anisaldehyde charring; R_(f) forCompound 1.16=0.54 and R_(f) for Compound 1.15=0.06 in EtOAc/Hexane(1:6)] to afford Compound 1.16 (15.5 g, 78.8% yield) as a thickcolorless liquid, which solidified slowly after 1-2 days at 0° C. Table5 describes the measured properties of the product.

TABLE 5 ¹H NMR δ = 5.20-5.15 (m, 1H), 5.03 (s, 1H), 2.86 (d, (500 MHz,CDCl₃) J = 10.0 Hz, 1H), 2.60 (d, J = 10.0 Hz, 1H), 2.46-2.28 (m, 5H),2.01 (s, 3H), 1.94-1.62 (m, 5H), 1.60-1.52 (m, 6H), 1.48-1.45 (m, 1H),1.41-1.36 (m, 4H), 1.20 (s, 3H), 0.82 (s, 3H) ¹³C NMR δ = 210.9, 170.5,147.2, 114.7, 70.0, 56.1, 55.6, (125 MHz, CDCl₃) 51.5, 47.4, 37.9, 35.9,34.4, 31.6, 30.7, 29.8, 26.4, 25.9, 25.5, 24.0, 23.5, 21.3, 17.9, 12.8Mass (m/z) 359.2 [M⁺ + 1], 376.2 [M⁺ + 18] IR (CHCl₃) 3421, 2928, 1734,1704, 1377, 1243 cm⁻¹ m.p. 88.5-91.2° C. [α]_(D) +30 (c = 1 in CHCl₃)

Example 5: Methyl (E)-3β-hydroxy-5β-chola-16(17),22(23)-diene-24-oateacetate (1.17)

Methyl propiolate (9.68 g, 114.95 mmol) is added to a solution ofCompound 1.16 (16.5 g, 46 mmol) in CH₂Cl₂ (220 mL) 0° C. The reactionmixture is warmed to ambient temperature and stirred for 1 h under aninert atmosphere. Ethyl aluminum dichloride (17.5 g, 137.8 mmol) isintroduced to the above mixture at 0° C. drop wise and the resultingreaction mass is again warmed to ambient temperature and stirredovernight. Upon completion of the reaction, as evidenced by TLC, thecrude reaction mixture is quenched with ice-water (100 mL) and theaqueous layer is extracted with EtOAc (3×150 mL). The combined organiclayer is washed with saturated brine solution (100 mL) and dried overMgSO₄ (50 g). The solvent is removed under vacuum and the crude materialis purified by column chromatography [49 (W)×600 (L) mm, 60-120 Meshsilica, 300 g] eluting with ethyl acetate/hexane (1:7) [15 mL fractions,10 mL/min elution, monitored by TLC and detected with either by UV light(254 nm) lamp or p-anisaldehyde charring; R_(f) for Compound 1.17=0.36and R_(f) for Compound 1.16=0.54 in EtOAc/Hexane (1:6)] to affordCompound 1.17 (16 g, 79% yield) as a colorless semi solid. Table 6describes the measured properties of the product.

TABLE 6 ¹H NMR δ = 6.89 (dd, J = 8.0, 16 Hz, 1H), 5.81 (d, J = 15 Hz,1H), 5.48 (s, (500 MHz, CDCl₃) 1H), 5.03 (s, 1H), 3.73 (s, 3H), 2.95 (t,J = 6.5 Hz, 1H), 2.45-2.36 (m, 2H), 2.30-2.17 (m, 2H), 2.04 (s, 3H),2.00-1.79 (m, 5H), 1.58 (s, 3H), 1.49-1.18 (m, 9H), 1.16 (s, 3H), 0.70(s, 3H) ¹³C NMR δ = 209.7, 170.1, 166.6, 154.4, 152.3, 124.5, 119.1,69.7, 56.3, (125 MHz, CDCl₃) 53.8, 52.3, 51.1, 49.8, 37.9, 35.7, 35.0,34.4, 30.5, 30.4, 29.6, 26.2, 25.6, 25.2, 23.3, 21.1, 19.2, 17.3 Mass(m/z) 443.0 [M⁺ + 1], 460.1 [M⁺ + 18] IR (CHCl₃) 3438, 2930, 1729, 1706,1653, 1448, 1435, 1243, 1022 cm⁻¹ [α]_(D) +59 (c = 1 in CHCl₃) HPLCpurity 94.4%; ret. time = 28.86, (Zorbax SB, C18; 250 × 4.6 mm, 5u),ACN: 5 mM TEA pH-2.5 with HClO₄ (Gradient); absorbance at 205 nm

Example 6: Methyl 3β-hydroxy-5β-cholan-11-one-24-oate acetate (1.18)

10% Pd/C (2.9 g, 20 wt %) is added to a solution of Compound 1.17 (14.5g, 32.8 mmol) in EtOAc (150 mL). The resulting slurry is hydrogenated ina Parr apparatus (50 psi) for 12 h. Upon complete disappearance ofstarting material, as evidenced by TLC [R_(f) for Compound 1.18=0.43 andR_(f) for Compound 1.17=0.43 in EtOAc/Hexane (1:3); however onlyCompound 1.17 is UV active from the conjugated ester chromophore], thecrude reaction mixture is filtered through a small plug of Celite andthe solvent is removed under vacuum to afford Compound 1.18 (14 g, 95.7%yield) as a colorless solid. Table 7 describes the measured propertiesof the product.

TABLE 7 ¹H NMR δ = 5.03 (s, 1H), 3.73 (s, 3H), 2.56 (d, J = 10 Hz, 1H),2.38-2.19 (500 MHz, CDCl₃) (m, 5H), 2.04 (s, 3H), 1.86-1.13 (m, 20H),1.12 (s, 3H), 0.86 (s, 3H), 0.62 (s, 3H) ¹³C NMR δ = 211.3, 174.3,170.5, 70.0, 58.3, 55.7, 55.0, 51.4, 50.8, 46.8, (125 MHz, CDCl₃) 37.9,36.7, 35.0, 34.3, 30.9, 30.7, 30.7, 29.7, 28.3, 26.6, 25.9, 25.5, 23.6,23.5, 21.4, 17.9, 12.7 Mass (m/z) 447.1 [M⁺ + 1], 464.1 [M⁺ + 18] IR(KBr) 3449, 2927, 1734, 1704, 1381, 1262, 1243 cm⁻¹ m.p. 174.2-175.7° C.(From CH₂Cl₂/Hexane) (Observed); 174.8-176.2° C. (Reported) [α]_(D) +39(c = 1 in CHCl₃)

Example 7: Methyl 3β-hydroxy-5β-chol-9(11)-ene-24-oate acetate (1.19)

PtO₂ (5.0 g, 100 wt %) is added to a solution of 1.18 (5.0 g, 11.2 mmol)in EtOAc (75 mL) in the presence of catalytic amount of AcOH (2.0 mL).The resulting slurry is hydrogenated in a Parr apparatus (70 psi) forabout 14-16 h. Upon completion of the reaction, the crude mixture isfiltered through a small plug of Celite and the solvent is removed undervacuum. The crude product is used for the elimination reaction withoutfurther purification.

SOCl₂ (1.98 g, 16.78 mmol) is introduced to a solution of the abovecrude material in pyridine (100 mL) drop wise at 0° C. The resultingreaction mixture is warmed to ambient temperature and stirred for about1 h. Upon completion of the reaction, as evidenced by TLC, pyridine isremoved under vacuum. The crude residue is diluted with ethyl acetate(100 mL) and washed with water (2×50 mL), saturated brine solution (100mL) and dried over MgSO₄ (40 g). The solvent is evaporated under vacuumand the crude material is purified by column chromatography [49 (W)×600(L) mm, 60-120 Mesh silica, 120 g] eluting with ethyl acetate/hexane(1:10) [10 mL fractions, 5 mL/min elution, monitored by TLC withp-anisaldehyde charring; R_(f) for Compound 1.19=0.51 and R_(f) forCompound 1.18=0.22 in EtOAc/Hexane (1:6)] to afford Compound 1.19 (4.1g, 85.4% yield) as a colorless solid. Table 8 describes the measuredproperties of the product.

TABLE 8 ¹H NMR δ = 5.33 (s, 1H), 5.03 (s, 1H), 3.66 (s, 3H), 2.37-2.32(m, 1H), (500 MHz, CDCl₃) 2.46-2.21 (m, 1H), 2.11-2.04 (m, 1H), 2.03 (s,3H), 1.99-1.10 (m, 22H), 1.07 (s, 3H), 0.92 (d, J = 7.0 Hz, 3H), 0.58(s, 3H) ¹³C NMR δ = 174.6, 170.6, 140.1, 118.9, 71.2, 56.1, 53.3, 51.3,42.0, 40.9, (125 MHz, CDCl₃) 38.9, 37.3, 36.4, 35.2, 31.9, 31.4, 31.0,30.9, 30.1, 28.2, 26.9, 26.2, 26.1, 25.3, 21.4, 17.9, 11.6 Mass (m/z)448.2 [M⁺ + 18] IR (KBr) 3447, 2935, 1735, 1379, 1261, 1245 cm⁻¹ m.p.188.6-191.2° C. (From CH₂Cl₂/Hexane) (Observed); 174-175° C. (Reported)[α]_(D) +37 (c = 1 in CHCl₃)

Example 8: Methyl 3β-hydroxy-5β-chol-9(11)-ene-12-one-24-oate acetate(1.20)

CrO₃ (8.0 g, 100 wt %, 80.0 mmol) is added to a solution of Compound1.19 (8.0 g, 18.6 mmol) in AcOH (150 mL). The resulting reaction mixtureis heated at 60° C. for about 24-36 h. Upon complete disappearance ofthe precursor, acetic acid is evaporated under vacuum, and the crudematerial is dissolved in diethyl ether (400 mL). The organic layer iswashed with water (2×100 mL), saturated brine solution (100 mL) anddried over MgSO₄ (40 g). The solvent is removed under vacuum and thecrude material is purified by column chromatography [49 (W)×600 (L) mm,60-120 Mesh silica, 120 g] eluting with ethyl acetate/hexane (1:5) [10mL fractions, 3 mL/min elution, monitored by TLC and detected with UVlight (254 nm) lamp; R_(f) for Compound 1.20=0.28 and R_(f) for Compound1.19=0.61 in EtOAc/Hexane (1:4)] to afford Compound 1.20 (5 g, 60.5%yield) as a colorless solid. Table 9 describes the measured propertiesof the product.

TABLE 9 ¹H NMR δ = 5.72 (s, 1H), 5.04 (s, 1H), 3.66 (s, 3H), 2.41-2.27(m, 3H), 2.03 (500 MHz, CDCl₃) (s, 3H), 1.94-1.58 (m, 9H), 1.48-1.30 (m,11H), 1.21 (s, 3H), 1.02 (d, J = 6.5 Hz, 3H), 0.91 (s, 3H) ¹³C NMR δ =205.1, 174.5, 170.4, 164.2, 123.1, 70.3, 53.4, 53.0, 51.3, 47.2, (500MHz, CDCl₃) 40.2, 37.7, 37.3, 35.2, 32.1, 31.4, 31.0, 30.6, 30.2, 27.3,26.5, 25.9, 25.6, 24.1, 21.3, 19.4, 10.6 Mass (m/z) 445.0 [M⁺ + 1],462.1 [M⁺ + 18] IR 3447, 2927, 2361, 2339, 1736, 1678, 1367, 1250 cm⁻¹m.p. 185.8-188.1° C. (From CH₂Cl₂/Hexane) [α]_(D) +62 (c = 1 in CHCl₃)HPLC purity 94.1%; ret. time = 23.89 (Hypersil BDS C18, 250 × 4.6 mm,5u, CH₃CN: 5 mM TEA, pH-2.5 with HClO₄ (Gradient); absorbance at 240 nm

Example 9: Methyl 3β-hydroxy-5β-cholane-12-one-24-oate acetate (1.21)

10% Pd/C (30 mg, 10 wt %) is added to a solution of Compound 1.20 (300mg, 0.675 mmol) in EtOAc (30 mL). The resulting slurry is hydrogenatedin a Parr apparatus (50 psi) for about 16 h. Upon complete disappearanceof starting material by TLC [R_(f) for Compound 1.21=0.44 and R_(f) forCompound 1.20=0.44 in EtOAc/Hexane (3:7); however only Compound 1.20 isUV active from its enone chromophore; additionally charring of Compound1.20 is faint but Compound 1.21 is bright], the crude reaction mixturewas filtered through a small plug of Celite and the solvent is removedunder vacuum to afford Compound 1.21 (270 m g, 90% yield) as a colorlesssolid. Table 10 describes the measured properties of the product.

TABLE 10 ¹H NMR δ = 5.04 (s, 1H), 3.64 (s, 3H), 2.52-2.47 (m, (500 MHz,CDCl₃) 1H), 2.38-2.25 (m, 3H), 2.23-2.03 (m, 2H), 2.02(s, 3H), 1.99-1.71(m, 8H), 1.49-1.11 (m, 12H), 1.05 (s, 3H), 1.01 (s, 3H), 0.85-0.84 (d, J= 7.0 Hz, 3H) ¹³C NMR δ = 214.7, 174.6, 170.5, 70.2, 58.7, 57.5, 51.4,(125 MHz, CDCl₃) 46.5, 43.7, 38.4, 36.9, 35.7, 35.6, 35.5, 31.3, 30.7,30.5, 27.5, 26.4, 25.9, 24.8, 24.3, 23.2, 21.4, 18.6, 11.7 Mass (m/z)447.0 [M⁺ + 1], 464.0 [M⁺ + 18] IR (KBr) 3447, 2935, 1735, 1704, 1260,1241 cm⁻¹ m.p. 179.6-182.7° C. (From CH₂Cl₂/Hexane) [α]_(D) +69 (c = 1in CHCl₃)

Example 10: Methyl 5β-chola-3,12-dione-24-oate (1.22)

NaOH (73 mg, 1.8 mmol) is added to a solution of Compound 2.1 (270 mg,0.6 mmol) in MeOH (10 mL). The resulting reaction mixture is stirred forabout 2 h at ambient temperature. Upon completion of the reaction, asevidenced by TLC, MeOH is removed under vacuum and the crude product isdiluted with ethyl acetate (20 mL). The organic layer is washed withsaturated brine solution (10 mL) and dried over MgSO₄ (5.0 g). Thesolvent is removed under vacuum and the crude material is used in theesterification reaction without purification.

SOCl₂ (0.1 mL, 1.35 mmol) is added drop-wise to a solution of the abovecrude material in MeOH (10 mL) 0° C. The resulting reaction mixture isstirred at ambient temperature for about 1 h. Upon completion of thereaction, MeOH is removed under vacuum. The crude reaction mixture isdiluted with EtOAc (30 mL), and the organic layer is washed with water(3×10 mL), saturated brine solution (15 mL) and dried over MgSO₄ (5 g).The solvent is evaporated under vacuum and the crude product is used forthe oxidation reaction without purification.

PCC (1.0 g, 4.6 mmol) is introduced in 3 equal portions to a solution ofthe obtained ester in CH₂Cl₂ (25 mL) over about 5 minutes. The resultingreaction mixture is stirred at ambient temperature for about 3-4 h. Uponcompletion of the reaction, as evidenced by TLC, the crude reactionmixture is filtered through a pad of Celite. The solvent is removedunder vacuum and the crude material is purified by column chromatography[19 (W)×400 (L) mm, 60-120 Mesh silica, 45 g] eluting with ethylacetate/hexane (1:6) [10 mL fractions, 5 mL/min elution, monitored byTLC with p-anisaldehyde charring; R_(f) for Compound 1.22=0.57 and R_(f)for Compound 1.21=0.71 in EtOAc/Hexane (2:3)] to afford Compound 1.22(170 mg, 70.8% yield) as a colorless solid. Table 11 describes themeasured properties of the product.

TABLE 11 ¹H NMR δ = 3.66 (s, 3H), 2.64-2.57 (m, 2H), 2.55-2.19 (m, 4H),2.17-2.02 (500 MHz, CDCl₃) (m, 3H), 1.99-1.21 (m, 17H), 1.11 (s, 3H),1.05 (s, 3H), 0.86 (d, J = 10.0 Hz, 3H) ¹³C NMR δ = 213.9, 211.8, 174.5,58.5, 57.5, 51.4, 46.5, 44.2, 43.7, 42.1, (125 MHz, CDCl₃) 38.3, 36.9,36.8, 35.6, 35.4, 31.3, 30.5, 27.4, 26.6, 25.4, 24.3, 22.1, 18.5, 11.7Mass (m/z) 403.1 [M⁺ + 1], 420.2 [M⁺ + 18] IR (KBr) 3457, 2925, 1737,1708, 1216, 1176 cm⁻¹ m.p. 133.7-135.9° C. (From CH₂Cl₂/Hexane) (Obs);136.5-137.5° C. (Rep) [α]_(D) +79 (c = 1 in CHCl₃)

Example 11: Methyl Deoxycholate (1.22-Ester)

LiAlH(O-^(t)Bu) (332 mg, 1.3 mmol) is introduced drop-wise to a solutionof Compound 1.22 (150 mg, 0.37 mmol) in THF (10 mL) under an inertatmosphere at ambient temperature. After being stirred for about 4-5 hr,the reaction mixture is quenched with Aqueous HCl (2 mL, 1N) and thecrude mixture is diluted with EtOAc (30 mL), washed with water (15 mL),saturated brine solution (10 mL) and dried over MgSO₄ (3 g). The solventis removed under vacuum, and the crude mass is purified by columnchromatography [29 (W)×500 (L) mm, 230-400 Mesh silica, 50 g] elutingwith MeOH/CH₂Cl₂ (1:20) [5 mL fractions, 3 mL/min elution, monitored byTLC with p-anisaldehyde charring; R_(f) for Compound 1.22-ester=0.42 andR_(f) for Compound 1.22=0.85 in MeOH/CH₂Cl₂ (1:9)] to afford methyldeoxycholate (Compound 1.22-ester) (110 mg, 72.8% yield) as a colorlesssolid. Table 12 describes the measured properties of the product.

TABLE 12 ¹H NMR δ = 3.97 (s, 1H), 3.65 (s, 3H), 3.63-3.59 (m, 1H),2.39-2.33 (m, (500 MHz, CDCl₃) 1H), 2.25-2.19 (m, 1H), 1.88-0.97 (m,24H), 0.95 (d, J = 6.0 Hz, 3H), 0.90 (s, 3H), 0.67(s, 3H) ¹³C NMR δ =174.7, 73.1, 71.7, 51.4, 48.2, 47.3, 46.5, 42.1, 36.4, 36.0, 35.2, (125MHz, CDCl₃) 35.1, 34.1, 33.6, 31.1, 30.9, 30.4, 28.6, 27.4, 27.1, 26.1,23.6, 23.1, 17.3, 12.7 Mass (m/z) 407.1 [M⁺ + 1], 424.2 [M⁺ + 18] IR(KBr) 3419, 2937, 2864, 1740, 1724, 1448, 1377, 1043 cm⁻¹ m.p.58.0-60.0° C. (under re-crystallization) [α]_(D) +36 (c = 1 in CHCl₃)

Example 11: Deoxycholic Acid

A solution of LiOH (23 mg, 0.55 mmol) in H₂O (2.0 mL) is added to asolution of 1.22-ester (110 mg, 0.27 mmol) in THF (4 mL). The resultingreaction mixture is stirred for about 2-3 h at ambient temperature. Upondisappearance of the ester by TLC [R_(f) for Compound DCA=0.35 and R_(f)for Compound 1.22-ester=0.42 in MeOH/CH₂Cl₂ (1:9)], the crude reactionmixture is diluted with ethyl acetate (10 mL) and triturated withsaturated brine solution to obtain a clear separation of the organiclayer. The organic layer was washed with saturated NH₄Cl solution (10mL), and dried over MgSO₄ (3.0 g). The solvent is removed under vacuumand any trace of water is removed by azetroping with toluene (3×5 mL) toafford deoxycholic acid (DCA) (100 mg, 94.3% yield) as a colorlesssolid. Table 13 describes the measured properties of the product.

TABLE 13 ¹H NMR δ = 3.77 (s, 1H), 3.38-3.33 (m, 1H), 2.20-2.15 (m, 1H),2.08-2.02 (500 MHz, DMSO) (m, 1H), 1.92-0.90 (m, 24H), 0.84-083 (d, J =5.0 Hz, 3H), 0.77 (s, 3H), 0.53 (s, 3H) ¹³C NMR δ = 175.9, 71.0, 69.9,47.4, 46.2, 45.9, 41.6, 36.3, 35.7, 35.1, 35.0, (125 MHz, DMSO) 33.8,32.9, 31.2, 31.2, 30.2, 28.6, 27.2, 26.9, 26.1, 23.5, 23.0, 16.9, 12.4Mass (m/z) 392 [M⁺, not detected], 410.2 [M⁺ + 18] IR 3445, 2931, 2867,1694, 1636, 1043 cm⁻¹ m.p. 173.2-175.5° C. (From THF/CH₂Cl₂) (Observed);174-176° C. (Reported, Alfa Aesar) and 171-174° C. (Reported, Aldrich)[α]_(D) +50 [c = 1 in MeOH and CHCl₃ (1:1)]; +54° (c = 2 in ethanol)[Alfa Aesar]

The yield of products for the exemplary processes described in Examples1 through 11 are described in Table 14.

TABLE 14 Overall Yield of Synthetic DCA Process MP (° C.) MP (° C.)Compound (observed) Reported % Yield Notes Hydrocortisone 1.13128.9-131.1 128.0-131.0 79.00 Hydrogenation, side chain cleavage, andPCC oxidation 1.14 176.6-180.5 Crude K-SELECTRIDE ® (potassiumtri-sec-butylborohydride) reduction 1.15 156.0-158.0 66.40 Acetylation1.16 88.5-91.2 78.80 Wittig 1.17 79.00 Ene 1.18 174.2-175.7 95.80Hydrogenation (Pd/C) 1.19 188.6-191.2 174.0-175.0 85.40 Thionylchloride/pyridine dehydration (2-step yield) 1.20 185.8-188.1 60.50Chromium trioxide allylic oxidation 1.21 179.6-182.7 90.00 Hydrogenation(Pd/C) 1.22 133.7-135.9 136.5-137.5 70.80 Hydrolysis, esterification,and oxidation 1.22-ester 58.0-60.0 72.80 LiAlH(O-^(t)Bu)₃ reduction DCA173.2-175.5 174.0-176.0 94.33 Hydrolysis of ester  7.00 Overall yield

Example 12: Comparison of Carbon Content of Synthetic DCA Versus BovineDCA

Synthetic DCA (three samples, prepared according to the methodsdisclosed above or according to WO 2008/157635 Examples 12-24 which isincorporated herein by reference in its entirety) and DCA purchased fromSIGMA (Sigma Aldrich PO Box 14508 St. Louis, Mo. 63178), PIERCE (PierceProtein Research Products PO Box 117, Rockford Ill. 61105 USA), and NZP(New Zealand Pharmaceuticals Limited, PO Box 1869, Palmerston North4440, New Zealand) were analyzed for their percent fossil carbon and ppt(parts per trillion) ¹⁴C carbon content. The percent fossil carbonprovides a measure of the amount of carbon in the molecule originatingfrom fossil fuel, which is expected to have very little ¹⁴C due to decayof this isotope. Intermediates used in the synthesis of DCA are derivedfrom fossil fuels. Conversely, DCA synthesized from animals is expectedto have approximately 1 ppt ¹⁴C and little to no fossil content. Theseexpectations are borne out in the analyses as shown in Table 15.(Radiocarbon analyses were carried out according to the American Societyfor Testing Materials ASTM D6866 procedure (ASTM international, 100 BarrHarbon Drive, PO Box C700, West Conshohocken, Pa. 19428-2959).

TABLE 15 Comparison of carbon content of DCA isolated from bovinesversus synthetic DCA DCA Fossil Carbon ppt C¹⁴ Bovine DCA (SIGMA)  0% 1ppt Bovine DCA (PIERCE)  0% 1 ppt Bovine DCA (NZP)  2% 1 ppt SyntheticDCA (sample 1) 13% 0.87 ppt Synthetic DCA (sample 2) 12% 0.88 pptSynthetic DCA (sample 3) 11% 0.89 ppt

Accordingly, in one embodiment provided is method for distinguishingsynthetic DCA from naturally derived DCA based on their fossil-derivedcarbon content and/or ¹⁴C content. In one aspect, provided is syntheticDCA having greater than 4% fossil-derived carbon. In another aspect,provided is synthetic DCA having less than 1 ppt ¹⁴C or less than 0.9ppt ¹⁴C.

What is claimed is:
 1. An aqueous pharmaceutical composition comprisingabout 0.1% w/v to about 2% w/v deoxycholic acid, or a pharmaceuticallyacceptable salt thereof, and at least one pharmaceutically acceptableexcipient and/or carrier, wherein the deoxycholic acid has a ¹⁴C contentof less than 1 ppt.
 2. The composition of claim 1, wherein thedeoxycholic acid has a ¹⁴C content of less than 0.9 ppt.
 3. Thecomposition of claim 1, wherein the deoxycholic acid has a ¹⁴C contentof 0.87 ppt.
 4. The composition of claim 1, wherein the deoxycholic acidhas a ¹⁴C content of 0.88 ppt.
 5. The composition of claim 1, whereinthe deoxycholic acid has a ¹⁴C content of 0.89 ppt.
 6. The compositionof claim 1 which is suitable for subcutaneous injection.